CN112088903A

CN112088903A - Application of macromolecular protein in antibacterial and antiviral disinfectant

Info

Publication number: CN112088903A
Application number: CN202011040661.9A
Authority: CN
Inventors: 朱小湘
Original assignee: Wuhan Yinzi Biological Technology Co ltd
Current assignee: Wuhan Yinzi Biological Technology Co ltd
Priority date: 2020-09-28
Filing date: 2020-09-28
Publication date: 2020-12-18

Abstract

The invention provides an application of macromolecular protein in an antibacterial and antiviral disinfectant, wherein the macromolecular protein is one or more of natural immune polypeptide and protein secreted by bacteria, fungi, animals or plants in the biological world, the disinfectant is applied to an in vitro environment, a body surface environment or an oral and nasal cavity environment, the macromolecular protein can stably exist in the in vitro environment and has lasting antivirus and sterilization activity, and the macromolecular protein is one or more of recombinant human RNases. The invention applies natural immune substances in the biological world to in-vitro disinfection products, realizes the inactivation of germs and viruses in the environment, and changes the traditional concept that natural immune protein is only used for medicines in human bodies.

Description

Application of macromolecular protein in antibacterial and antiviral disinfectant

Technical Field

The invention relates to the technical field of disinfection products, in particular to application of macromolecular protein in an antibacterial and antiviral disinfectant, and specifically relates to application of natural immune molecules RNases in a human body to in-vitro sterilization and disinfection, prevention of germ infection or reduction of germ carrying capacity in the environment and alleviation of infection.

Background

Human antiviral and antibacterial depends on the immunological competence of human body, including innate immunity and acquired immunity after infection-vaccine. Innate immunity includes cellular immunity and humoral immunity. Innate humoral immunity is composed of various polypeptides, immune factors and certain active proteins in the body that are resistant to bacterial, fungal and viral infections. Some of these ribonucleases (RNases) protect against invading microorganisms by secreting enzymes from liquids such as tears, saliva, mucous and sweat. The RNase a superfamily is a vertebrate-specific family of proteins that encompasses eight functional members of the human family. These proteins are secreted by a variety of innate immune cells from bone marrow cells to epithelial cells, and their levels in our body are associated with infectious and inflammatory processes due to the important role of the secreted RNase in the extracellular space of the body.

The RNase A superfamily consists of eight enzymatically active, "standard" ribonucleases that are structurally homologous to RNase A. They are secreted proteins having a disulfide-bonded tertiary structure and capable of degrading RNA (ribonucleic acid). In humans and mice, all genes are encoded in a tight region of chromosome 14 and are thought to originate from gene replication. Although the gene contains multiple exons, the coding region is contributed by a single exon. Their physiological effects are important for host defense, angiogenesis and digestion. The first member is RNase 1, which is produced not only in pancreatic enzymes but also in various cells including vascular endothelial cells, and after secretion, it degrades vascular poly-RNA and has anti-HIV-1 activity.

Rnases

2 and 3 are eosinophil-secreted proteins, known as eosinophil-derived neurotoxin (EDN) and Eosinophil Cationic Protein (ECP), respectively, which both have anti-RNA viral effects. RNase 4 is present in a variety of tissues. RNase 5, also known as angiogenin, induces blood vessel growth. RNase7 is the most abundant RNase in the skin, while RNase 8 is expressed in the placenta. They are involved in various physiological and pathological processes, have destructive and inactivating effects on bacteria and viruses, and are important members of natural immune defense in the biological world.

Although RNases play an important role in innate immunity in humans, recombinant RNases have very few applications in the treatment and prevention of diseases other than biomedical research, and in particular, have no applications in the prevention and disinfection of microbial infections and infectious diseases. This gap is mainly due to the fact that the research on the physicochemical stability of these RNases in the prior art is very little, the knowledge is not enough, and the common sense concept of protein properties is that the enzyme protein needs body temperature (37 ℃) to exert the function and activity of the enzyme, and the protein is unstable in the natural environment, is easy to denaturize, putrefy and degrade, but cannot persist in vitro, kill pathogenic microorganisms, and play a role in preventing diseases.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides the application of macromolecular polypeptide protein in an antibacterial and antiviral disinfectant, natural immune polypeptide and protein secreted in the biological world are screened, macromolecular protein which can stably exist in an in vitro environment and has lasting activity is found, and the protein is applied to a disinfection product, so that the inactivation of pathogenic bacteria and viruses in the environment is realized, and the traditional concept that the natural immune protein is only used for medicines in a human body is changed.

The technical scheme adopted by the invention is as follows:

the application of a macromolecular protein in an antibacterial and antiviral disinfectant, wherein the macromolecular protein is one or more of natural immune polypeptide and protein secreted by bacteria, fungi, animals or plants in the biological kingdom. The macromolecular protein has stable structure and certain activity in an in-vitro normal-temperature environment.

Preferably, the disinfectant acts in an in vitro environment, a body surface environment or an oronasal environment.

Preferably, the macromolecular protein is one or more of RNaseA family proteins.

More preferably, the macromolecular protein is one or more of RNase2, RNase3 and RNase 7; the macromolecular protein is human protein, and the human immune polypeptide-protein molecule has less harm to human body, thereby avoiding causing anaphylactic reaction of human body.

The preparation method of the macromolecular protein comprises the following steps: directly extracting from organisms, preparing protein by adopting a recombination technology, or synthesizing the protein in vitro by physical, chemical and biological technologies; the method for preparing the macromolecular protein by adopting the recombinant technology comprises the following steps: protein expressed and purified by prokaryotic cells or eukaryotic cells through recombinant DNA, wherein the purity is more than 90 percent, and the concentration is 1-20 mu M; the eukaryotic cell comprises a fungal cell, an insect cell, an animal cell or a human cell; specifically, the prokaryotic cell can be escherichia coli (e.coli), and the eukaryotic cell can be human 293 cell.

Preferably, the disinfectant is a liquid preparation, or the disinfectant is a dry powder, and the dry powder is mixed with a solvent in an action environment to play a role; more preferably, the solvent of the disinfectant is water.

Preferably, the concentration of the macromolecular protein in the disinfectant is 0.01-5000 nM.

Preferably, the disinfectant also contains a protein stabilizer, a buffering agent and a preservative.

In the above technical solution, the protein stabilizer includes one or more of preservative and antibacterial protein stabilizers, protein and amino acid protein stabilizers, antioxidant protein stabilizers and carbohydrate protein stabilizers. Specifically, the preservative and antibacterial protein stabilizer comprises sodium azide, glycerol, benzalkonium chloride, benzalkonium bromide and thymol; the protein and amino acid protein stabilizers include beta-alanine, L-serine, N-dimethylglycine, L-isoleucine, taurine, glycine, albumin, L-arginine, sarcosine, L-cystine, L-proline, L-glutamic acid, and L-lysine; the antioxidant protein stabilizer comprises trihydroxypropyl phosphine, tris (2-carboxyethyl) phosphine hydrochloride, ethylene diamine tetraacetic acid, DL-dithiothreitol and 2-mercaptoethanol; the carbohydrate protein stabilizer comprises alpha-lactose monohydrate, D-sorbitol and D- (+) -trehalose dihydrate. The pH value of the phosphoric acid buffering agent is 7-8, and the preservative is benzalkonium chloride or/and potassium sorbate.

The invention has the beneficial effects that:

(1) the invention uses natural immune macromolecular protein with sterilization and disinfection in the biological world as an in vitro antibacterial and antiviral disinfectant, and the macromolecular protein can stably exist in the in vitro environment and has the function of inhibiting the virus activity independently or in combination with other substances; changes the industrial prejudice that the natural immune protein is only used for medicines in human bodies.

(2) The disinfectant provided by the invention contains one or more than 8 recombinant human-derived RNases as an active ingredient, wherein the RNases are natural secreted proteins produced by human bodies, and the molecular weight of the secreted proteins is about 130 amino acids. The existing research shows that the compounds have the functions of resisting bacteria and viruses and regulating the immunity of organisms in human bodies; experiments prove that the compounds have strong stability and activity in an in vitro environment and can be used for killing germs and viruses in the environment.

(3) Among recombinant human-derived RNases, RNase2, RNase3 and RNase7 have the best stability and activity: RNase2, RNase3 and RNase7 all have RNase activity at 0 ℃ to 80 ℃; the RNase2 and the RNase3 are heated to 100 ℃ and denatured for 10-30 minutes, and after renaturation at room temperature, the activity is not obviously different from that before denaturation; RNase2 and RNase3 are stored for 3 days to 3 months, proteins are not degraded, and the activity is not obviously different; after RNase2 and RNase3 were exposed to the natural environment, several good RNase activities were recovered after 72 h.

(4) The disinfectant containing the recombinant humanized RNases is suitable for various environmental climate temperature conditions, can be used on the surfaces of bodies, oral cavities, nasal cavities, wounds and the surfaces of air and solid matters in the environment, and can eliminate and kill more than 90 percent of germs and viruses; the disinfectant has lasting activity and cumulative concentration and dose-effect of enhancing enzyme molecules after being continuously used for multiple times in a short period.

(5) Compared with the existing small molecule disinfectant, the disinfectant has the advantages of long acting time, high efficiency, no stimulation to human body, environmental protection, cyclic effect and the like; moreover, it can be used in places where people gather, such as restaurants, supermarkets, cinemas, buses, offices, outpatient wards and the like in use, and has no adverse side effects such as smell, irritation, toxicity, corrosion and the like. The 'exogenous' RNase can even act on the body surface, oral and nasal cavities, and even in the trachea, alveoli and esophageal intestines (the natural environment of RNase).

(6) The disinfectant is especially suitable for the elderly, people with congenital hypoimmunity due to other chronic diseases, and places where the people are.

Drawings

FIG. 1 is a graph of the in vitro activity of recombinant RNases as a function of temperature;

FIG. 2 is a gel electrophoresis of RNA degradation by recombinant RNase2 in disinfectant after heat denaturation and renaturation;

FIG. 3 is a SDS-PAGE pattern of recombinant RNase in the disinfectant under different storage conditions;

FIG. 4 is a gel electrophoresis of RNA degraded by recombinant RNase in disinfectant solution stored for 3 months;

FIG. 5 is a gel electrophoresis of RNA degraded by recombinant RNase2 and recombinant RNase3 in disinfectant solutions at different exposure times;

FIG. 6 is a graph comparing the inhibition of the recombinant RNase7 on different bacteria and fungi.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The experimental methods in the present invention are conventional methods unless otherwise specified. The experimental materials used in the present invention were all purchased from the market unless otherwise specified.

Example 1

An antibacterial and antiviral disinfectant for macromolecular proteins, said disinfectant comprising one or more of recombinant human RNase enzyme family proteins.

The entries of the record of human RNases in the Uniprot database are shown in the following table:

these proteins have an enzymatic role, they catalyze the following reactions:

An(RNA)containing cytidine+H(2)O＝an(RNA)-3'-cytidine-3'-phosphate+a 5'-hydroxy-ribonucleotide-3'-(RNA)An(RNA)containing uridine+H(2)O＝an(RNA)-3'-uridine-3'-phosphate+a 5'-hydroxy-ribonucleotide-3'-(RNA)

these proteins are naturally distributed outside the cells of various organs and tissues of the whole body, and are secreted to the body surface, oral cavity, intestinal tract and urethra through tears, saliva, mucus and sweat, and are used as natural immune molecules to resist pathogenic microorganisms in the human body.

The RNases in the embodiment are recombinant humanized RNases prepared by adopting a recombinant technology, and the specific preparation method comprises the following steps: coli (escherichia coli), Sf9 insect cells or 293 human cells through recombinant DNA expression and purification, and the purity of the protein is more than 90%, and the concentration is 1-200 muM. The preparation of recombinant RNases is prior art and will not be described herein in any greater detail.

The recombinant RNases in this embodiment are specifically one or more of RNase2, RNase3 and RNase 7.

The disinfectant is a liquid preparation, water is used as a solvent, and the concentration of macromolecular protein is 0.01-5000 nM.

In order to maintain long-term activity and improve the using effect of the disinfectant, a protein stabilizer, a phosphate buffer and a preservative can be added into the disinfectant. The protein stabilizer comprises one or more of antiseptic and antibacterial protein stabilizers, protein and amino acid protein stabilizers, antioxidant protein stabilizers and carbohydrate protein stabilizers. The pH value of the phosphate buffer is 7-8, and the phosphate buffer comprises the following specific components: 137mM NaCl, 2.7mM KCl, 8mM Na₂HPO₄And 2mM KH₂PO₄. The antiseptic is used for preventing growth of microorganism and has antibacterial and disinfectant effects, and is specifically benzalkonium chloride or benzalkonium chlorideAnd potassium sorbate.

Viruses that can be inhibited by the above disinfectant in vitro or on body surface include, but are not limited to, the following: preventing respiratory syncytial virus, all influenza virus, parainfluenza virus, rhinovirus, poliovirus, coxsackie virus, dengue fever virus, rotavirus, SARS virus, MERS virus, Ebola virus (Ebola virus), Marburg virus, novel coronavirus (SARS-CoV-2) and other respiratory infectious viruses, or AIDS virus (which is retrovirus), hepatitis C virus and encephalitis B virus.

Because the active ingredients are recombinant RNases, and the types and the dosage of the preservative, the stabilizer and the solvent are adjusted, the disinfectant can be applied to any scenes, such as food, daily necessities, body surfaces and the like, and can even enter the mouth, nose, eyes and other parts of a human body in an atomized state (in the form of aerosol) to reduce viruses.

It should be noted that the recombinant RNases used in the present invention are both prepared by the applicant itself according to the prior art and partially purchased from several companies, and the properties of RNase2 and RNase3 were not determined for a certain product and were not accidental.

EXAMPLE 2 stability of disinfectant

(1) Thermostability of recombinant RNases

Three RNases were tested for activity changes under various temperature conditions. The detection process of the RNase activity is specifically as follows: catalytic reaction 40mg of yeast tRNA was added as substrate to 0.8mL of reaction solution (containing 40mM sodium phosphate, rhRNase2, or rhRNase3, or rhRNase7, or buffer control, pH 7.0 and 10 mL). The reaction was stopped at a given time point by adding 40nM lanthanum nitrate to ice-cold 3% perchloric acid. After that, the solution was centrifuged, and the remaining acid-soluble ribonucleotides in the supernatant were quantitatively analyzed at 260nM by spectrophotometry. All time points were tested in triplicate. The calculation includes the following approximations: the average molecular weight (Mr) of tRNA was 28,100(75-90 ribonucleotides/tRNA molecule 1Mr 341/ribonucleotide), and the absorbance at 260nm was 1.0, corresponding to 40mg RNA. The RNase activity measured at 0 ℃ was taken as 100%, and the RNase activity measured at each subsequent temperature point was converted into percentages with respect to the RNase activity measured at 0 ℃.

As shown in FIG. 1, the RNase2, RNase3 and RNase7 all had good activity from 0 ℃ to 80 ℃ and the activity increased with increasing temperature to 50 ℃ and then decreased with increasing temperature. The RNases have good activity function in the daily living environment temperature of human beings.

(2) Renaturation of recombinant RNases

Heating disinfectant containing RNase2 (RNase2 concentration is 2nM) to 100 deg.C, denaturing for 20min, and renaturing at room temperature to degrade total RNA of eukaryotic cells. The results are shown in FIG. 2, in which

lanes

1, 2 and 3 show that the total RNA of eukaryotic cells not digested by RNase2 was left at room temperature for 10 minutes,

lanes

4, 5 and 6 show that the total RNA of eukaryotic cells was digested by RNase2 at room temperature (25 ℃) for 10 minutes,

lanes

7, 8 and 9 show that the total RNA was digested by RNase 2100 ℃ (after denaturation) for 10 minutes, and

lanes

10, 11 and 12 show that the total RNA was digested by RNase2 after heat denaturation and renaturation at room temperature (25 ℃) for 10 minutes. As can be seen, the RNA in

lanes

1, 2, 3 and

lanes

7, 8, 9 was not degraded, indicating that RNase2 heated to 100 ℃ lost the function of degrading RNA; the RNA in

lanes

4, 5, and 6 and

lanes

10, 11, and 12 has been degraded, indicating that RNase2 in the disinfectant has the function of degrading RNA, and RNase2 after heat denaturation can restore the structure and function by itself.

(3) Storage stability of disinfectant

Storing disinfectant solution containing RNase2 or RNase3, wherein the concentration of RNase2 or RNase3 is 2-50nM at different temperatures, storing for a period of time, concentrating, and detecting, wherein the sample addition amount of each lane is about 1 μ g.

As shown in FIG. 3, lanes 1-6 show the RNase2 stored at-80 deg.C, -20 deg.C, 4 deg.C, 25 deg.C, 37 deg.C, and-80 deg.C for 3 months, respectively; lanes 7-11 show RNase3 at-20 deg.C, 4 deg.C, 25 deg.C, 37 deg.C, and-80 deg.C for 3 months. As can be seen from the figure, the proteins of RNase2 and RNase3 were not degraded after 3 months of storage at the above temperature, and there was no significant difference in activity.

Respectively storing disinfectant containing RNase2 or RNase3 at different temperatures for 3 months, and then digesting total RNA of eukaryotic cells, wherein the reaction conditions are as follows: RNase in an amount of about 0.5-1. mu.g at 37 ℃ for 30 min.

The results are shown in FIG. 4, in which the recombinant RNase2 used in lanes 1-6 was RNase2 stored at-80 ℃, 20 ℃, 4 ℃, 25 ℃, 37 ℃ and 80 ℃ for 3 months in this order; RNase3 used in lanes 7-11 was RNase3 stored at-20 deg.C, 4 deg.C, 25 deg.C, 37 deg.C, and-80 deg.C for 3 months in this order. As can be seen, there was no significant difference in the function of degrading RNA between the recombinant RNase2 and the recombinant RNase3 after long-term storage at the above-mentioned temperatures.

The detection results show that the recombinant RNase2 and the recombinant RNase3 in the disinfectant can be stored for a long time at a common temperature (-20-37 ℃) and the storage life is more than 3 months.

(4) Activity of disinfectant liquid exposed to natural environment

mu.L of 5nM recombinant RNase2 or recombinant RNase3 disinfectant was pipetted onto the surface of histological glass slides, placed openly in 10-person 30-square-meter office space, and RNases were recovered after 0, 0.5, 2, 8, 24 and 72 hours. The total RNA of eukaryotic cells was added to the recovered tube, reacted at 37 ℃ for 1 hour, and then subjected to gel electrophoresis.

The detection result is shown in FIG. 5, lane R is total undigested eukaryotic cell RNA;

lanes

1, 3, 5, 7, 9 and 11 are total RNA digested with recombinant RNase2 at different exposure times, respectively, and the exposure time of recombinant RNase2 was 0, 0.5, 2, 8, 24 and 72 hours in this order;

lanes

2, 4, 6, 8,10, 12 are total RNA digested with recombinant RNase3 at different exposure times, and recombinant RNase3 was exposed for 0, 0.5, 2, 8, 24, 72 hours; lane C is total eukaryotic RNA digested by washing the blank glass slide exposed for 72 hours with the recovery solution.

The recombinant human RNases can naturally form 4 disulfide bonds, and the structure and the function of the RNase are stabilized. Previous experiments have demonstrated that RNase2 and RNase3 can be stored at 37 ℃ for more than 3 months with good protein integrity and function. However, the RNase function was reduced after the exposure for 24-72 hours in this experiment. This reduction may be due to the RNases drifting with the air stream after the exposed aqueous solution evaporates, or may be degraded in small portions by various physical, chemical, and biological factors in the environment, but the drifting and residual RNase activity may still be present. Thus, these RNases remain intact in the environment for a longer period of time and act as virucidal agents.

EXAMPLE 3 bacteriostatic action of disinfectant

After incubating clinically common pathogenic bacteria and fungi in 100. mu.l 10mM sodium phosphate buffer (pH 7.4) containing 1% tryptone in soy broth and RNase7 at various concentrations above, at 37 ℃ for 3 hours, the cells were transferred to a petri dish overnight and colony Counts (CFU) were calculated the next day, and CFU/mL was calculated.

The results of the measurements are shown in FIG. 6, which are a statistical chart of Enterobacter (Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Propionibacterium acnes (Propionibacterium acnes), Enterococcus Faecium (Enterococcus faecalis), and Candida albicans (Candida albicans) in this order; as can be seen, RNase7 has an inactivating effect on all these pathogens, and the various pathogens have different sensitivities to RNase, with the highest sensitivity to enterococci and the lowest sensitivity to Candida albicans (fungi). It is noteworthy that the number of all pathogens in the experiment was high, far above the usual pathogenic load.

EXAMPLE 4 Virus inhibition by disinfectant

(1) Antiviral Capacity of RNase

The response of human eosinophils (0.4X 106/mL) to reduced infectivity of respiratory syncytial virus (RSV-A) and parainfluenza virus (PIV) types 1, 2, 3 with or without ribonuclease inhibitor (RI, 200U/mL) is shown in the following Table:

by adding an RNase inhibitor, it was demonstrated that the antiviral effect of eosinophils is due to the action of RNase.

(2) Comparison of the inactivation Capacity of RNase2 for different RNA viruses

The number of active viruses was determined by treating the same amounts of virus 1 and virus 2 with different doses of recombinant RNase2 and infecting the cultured cells with the treated viruses, as shown in the following table (all experiments were repeated 3 times, 2 parts each, results are mean,. + -. standard deviation):

as can be seen from the above table, the number of active viruses decreased with increasing amounts of RNase2, demonstrating that RNase2 has inhibitory effect on both viruses, but the viruses have different susceptibility to RNase 2. The reason for this is presumed to be: both the respiratory syncytial virus and the parainfluenza virus have Matrix Proteins below a double-layer lipid membrane, are key Proteins consisting of virus structures, and play an important role in assembling the virus and protecting virus outer membranes and genomes; respiratory syncytial virus has a different Matrix protein than parainfluenza virus, 195 and 353 amino acids, respectively, and may result in a different antiviral efficacy of RNase 2.

The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. The application of the macromolecular protein in the antibacterial and antiviral disinfectant is characterized in that the macromolecular protein is one or more of natural immune polypeptide and protein secreted by bacteria, fungi, animals or plants in the biological world, and the disinfectant acts on an in vitro environment, a body surface environment or an oral and nasal cavity environment.

2. The use according to claim 1, wherein the macromolecular protein is one or more of the RNase family of enzymes proteins.

3. The use according to claim 2, wherein the macromolecular protein is one or more of RNase2, RNase3 and RNase 7.

4. The use according to claim 3, wherein the macromolecular protein is a human protein; the preparation method of the macromolecular protein comprises the following steps: directly extracting from organisms, preparing protein by adopting a recombination technology, or synthesizing the protein in vitro by physical, chemical and biological technologies.

5. The use according to claim 4, wherein the recombinant production of the macromolecular protein is carried out by: protein expressed and purified by prokaryotic cells or eukaryotic cells through recombinant DNA, and the purity is more than 90 percent; the eukaryotic cell includes a fungal cell, an insect cell, an animal cell, or a human cell.

6. Use according to claim 2, wherein the disinfectant is a liquid formulation or the disinfectant is a dry powder formulation and the dry powder formulation is worked in admixture with a solvent in the working environment.

7. Use according to claim 5, wherein the solvent of the disinfectant is water.

8. The use according to claim 2, wherein the concentration of the macromolecular protein is 0.01 to 5000 nM.

9. The use of claim 2, wherein the disinfectant further comprises a protein stabilizer, a buffering agent, and a preservative.

10. The use of claim 9, wherein the protein stabilizing agent is one or more of sodium azide, glycerol, benzalkonium chloride, benzalkonium bromide, thymol, β -alanine, L-serine, N-dimethylglycine, L-isoleucine, taurine, glycine, albumin, L-arginine, sarcosine, L-cystine, L-proline, L-glutamic acid, L-lysine, trishydroxypropyl phosphine, tris (2-carboxyethyl) phosphine hydrochloride, ethylenediaminetetraacetic acid, DL-dithiothreitol, 2-mercaptoethanol, α -lactose monohydrate, D-sorbitol, D- (+) -trehalose dihydrate.